Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC. 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of alpha-amylases referred to as “TERMAMYL®-like alpha-amylases” and variants thereof are known from, e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874. TERMAMYL®-like alpha-amylases are very thermostable and therefore suitable for processes carried out at high temperatures such as starch liquefaction in dextrose production processes.
Another group of alpha-amylases are referred to as “FUNGAMYL™-like alpha-amylases”, which are alpha-amylases related or homologous to the alpha-amylase derived from Aspergillus oryzae. The FUNGAMYL™-like alpha-amylases have a relatively low thermostability the commercial product sold under the tradename FUNGAMYL™ by Novozymes A/S, Denmark, has a optimum around 55° C., and is not suitable for processes carried out at high temperatures. FUNGAMYL™-like alpha-amylases are today used for making syrups for, e.g., the brewing industry.
Clearly, it would be advantageous to provide an alpha-amylase with increased thermostability preferably at an acidic pH. This is no new realization, but actually a very long-felt need in the art. As far back as in 1980, Somkuti and Steinberg described a thermoacidophilic extracellular alpha-amylase of Rhizomucor pusillus (Mucor pusillus), that they managed to isolate and characterize. They state that: “Since high temp and acidic pH are optimum conditions for the economic hydrolysis of starch, the use of thermostable and acid-stable amylases of microbial origin for industrial purposes has been recommended”, and go on to conclude about the Rhizomucor amylase that: “It is apparently the first example of fungal alpha-amylase exhibiting both acidophily and thermophily simultaneously. Consequently, the alpha-amylase of M.pusillus should be of economic importance.” (Somkuti and Steinberg, 1980, “Thermoacidophilic extracellular amylase of Mucor pusillus”, Dev. Indust. Microbiol. 21:327-337).
However, despite the very clear conclusions by Somkuti and Steinberg back in 1980, the gene encoding the Rhizomucor pusillus alpha-amylase had until today not been cloned or sequenced, and the amylase had until today not been produced recombinantly in industrially relevant amounts. In 1987 an improved purification method was reported, but still only for enzyme produced by the wild-type Rhizomucor pusillus (Turchi and Becker, 1987, Curr. Microbiol. 15:203-205).